The present invention relates to displacement chromatography of oligonucleotides using low molecular weight, high affinity anionic displacers.
Oligonucleotides have generated significant interest as drug candidates for a wide variety of diseases, in particular as antisense therapeutics and as potent antibiotics. Several antisense oligonucleotide drugs are currently undergoing human clinical trials, and many others are in a preclinical phase.
Oligonucleotides are single strands of nucleic acids with DNA or RNA bases, ranging in length from two to 50 bases or nucleotides. Phosphorothioate derivatives of oligonucleotides have also been utilized because of their higher in vivo stability compared to the parent phosphodiester compounds. In the phosphorothioate derivatives, a non-bridging oxygen atom in the phosphodiester backbone is replaced with a sulfur atom. This substitution enhances nuclease resistance and thus, in vivo stability.
Solid-phase synthesis methods are now available for large-scale preparation of oligonucleotides. Solid-state synthesis of an oligonucleotide results in a crude product containing not only the desired full-length (n-length or n-mer) oligonucleotide, but also multiple, closely-related deletion or xe2x80x9cfailure sequences,xe2x80x9d primarily of length nxe2x88x921. These so-called failure sequences arise by failure to add a base at the necessary position. Such failures or deletions can arise at multiple positions along the chain. Multiple failure sequences, of length (nxe2x80x941, nxe2x88x922, nxe2x88x92x), are also present for any given length. In addition, (n+1)mers may be present. Therefore, purification methods that operate on a preparative scale are needed.
Chromatographic preparation and purification of oligonucleotides can potentially provide the necessary scale and purity, but can also present unique challenges. First, oligonucleotides exhibit an extremely high binding affinity for anion-exchange chromatographic resins as compared to molecules typically encountered in biopharmaceutical purification (for example, proteins). Second, the failure sequences present are so closely-related to the desired product that the components are difficult to separate. Finally, oligonucleotides exhibit several centers of isomerism leading to the possibility of considerable heterogeneity of the mixtures of product and failure sequences.
A chromatographic system can be operated in one of two major modes, elution (including linear gradient, step gradient, and isocratic elution) or displacement. The two modes may be distinguished both in theory and in practice. In elution chromatography, a solution of the sample to be purified is applied to a stationary phase, commonly in a column. A mobile phase is chosen such that the sample is neither irreversibly adsorbed nor totally unadsorbed, but rather binds reversibly. As the mobile phase is caused to flow over the stationary phase, an equilibrium is established between the mobile phase and the stationary phase whereby components of the sample pass along the column at speeds which reflects their affinity for the stationary phase relative to the other components that may occur in the original sample. The differential migration process is outlined schematically in FIG. 1, and a typical chromatogram is shown in FIG. 2. Of particular note is the fact that the eluting solvent front, or zero column volume in isocratic elution, always precedes the sample off the column.
A modification and extension of isocratic elution chromatography is found in step gradient chromatography wherein a series of eluants of varying composition are passed over the stationary phase.
In ion-exchange chromatography, step changes in the mobile phase salt concentration and/or pH are employed to elute or desorb materials such as, for example, proteins.
A schematic illustrating the operation of a chromatographic system in displacement mode is shown in FIG. 3. The column is initially equilibrated with a buffer in which most of the components to be separated have a relatively high affinity for the stationary phase. Following the equilibration step, a feed mixture containing the components to be separated is introduced into the column and is then followed by a constant infusion of the displacer solution. A displacer is selected such that it has a higher affinity for the stationary phase than any of the feed components. As a result, the displacer can effectively drive the feed components off the column ahead of its front. Under appropriate conditions, the displacer induces the feed components to develop into adjacent xe2x80x9csquarewavexe2x80x9d zones of highly concentrated, often pure material. The displacer emerges from the column following the zones of purified components. After the breakthrough of the displacer with the column effluent, the column is regenerated and is ready for another cycle.
An important distinction between displacement chromatography and elution chromatography is that in elution chromatography, desorbents, including salts for ion-exchange chromatography, move through the feed zones, while in displacement chromatography, the displacer front always remains behind the adjacent feed zones in the displacement train. This distinction is important because relatively large separation factors are generally required to achieve satisfactory resolution in elution chromatography, while displacement chromatography can potentially purify components from mixtures having low separation factors.
A key operational feature which distinguishes displacement chromatography from elution chromatography is the use of a displacer molecule. In elution chromatography, the eluant usually has a lower affinity for the stationary phase than any of the components in the mixture to be separated, whereas in displacement chromatography, the eluant, which is the displacer, has a higher affinity.
Displacement chromatography has some particularly advantageous characteristics for process scale chromatography of biological macromolecules such as oligonucleotides. First, displacement chromatography can concentrate components from mixtures. By comparison, isocratic elution chromatography results in product dilution during separation. Second, displacement chromatography can achieve product separation and concentration in a single step. Further, since the displacement process operates in the nonlinear region of the equilibrium isotherm, high column loadings are possible. This allows for improved column utilization compared to elution chromatography. Furthermore, displacement chromatography can purify components from mixtures having low separation factors, while relatively large separation factors are required for satisfactory resolution in desorption chromatography.
Preparative ion-exchange chromatography operated in the displacement mode is, therefore, a potentially attractive method for purifying oligonucleotides because of the high resolution and high throughput that can be obtained. However, displacement chromatography, as it is traditionally known, has a number of drawbacks compared to elution chromatography for the purification of oligonucleotides. Two of the major problems are difficulty in regeneration of the column and the presence of displacer in some of the purified fractions.
Since the displacement process uses a high affinity compound as the displacer, the time for regeneration and re-equilibration can be long compared to elution chromatography. The second problem, that of contamination by the displacer, has arisen because a common characteristic of displacers used in ion-exchange systems has been their relatively high molecular weight. Heretofore the art has taught the use of high molecular weight polyelectrolyte displacers on the assumption that it is necessary to have a large polyelectrolyte in order to ensure a higher binding coefficient than the biomolecule that is to be displaced. The rationale behind such an assumption is that the binding of a molecule to an adsorbent surface of an ion-exchange stationary phase is related only to its characteristic charge. Characteristic charge is the average number of sites of interaction of a solute with a stationary phase. High molecular weight displacers exhibit both of the disadvantages enumerated above: they bind tightly to the stationary phase and, therefore, require stringent conditions for regenerating the column, and traces of the displacer that may contaminate the product fraction are difficult to remove.
Low molecular weight displacers for protein separation that do not require extensive regeneration of the column and that can be readily removed from the product have been described by Cramer et al. (U.S. Pat. No. 5,606,033, issued Feb. 25, 1997; U.S. Pat. No. 5,478,924, issued Dec. 26, 1995). However, while the low molecular weight displacers identified so far have been successful in displacing moderately bound biomolecules such as proteins, they have been unsuccessful in displacing very highly retained compounds in ion-exchange systems, for example, oligonucleotides. Prior art disclosures relating to purification of oligonucleotides in ion-exchange systems have described only the use of relatively large polyelectrolytes ( greater than 40,000 Daltons) as displacers. For example, use of displacement chromatography with a high molecular weight polyelectrolyte displacer, a sulfonated polysaccaride, for the purification of oligonucleotides has been reported by Gerstner et al. (J. Nucl. Acids Res. 1995, 23, 2292-2299).
Therefore, there is a need for a separation process of oligonucleotides having the advantages of displacement chromatography (high resolution and high throughput) while avoiding the disadvantages (difficult column regeneration and contamination of the product with the displacer.)
In one aspect, the present invention relates to methods for purifying an oligonucleotide, or several oligonucleotides, comprising:
(a) loading said oligonucleotide onto an anion-exchange column having a stationary phase; and
(b) displacing said oligonucleotide from said anion-exchange column by an anionic displacer having a molecular weight of less than about 10,000, said anionic displacer having a higher affinity for said stationary phase of said anion-exchange column than said oligonucleotide.
Preferably, the molecular weight of the displacer is less than about 5,000. More preferably, the molecular weight of the displacer is less than about 2,500.
A preferred composition for the displacer is a substituted or unsubstituted aromatic compound having at least one anionic substituent. Another preferred composition for the displacer is a substituted or unsubstituted aliphatic compound having at least one anionic substituent. Preferred anionic substitutents are sulfate, sulfonate, phosphate, phosphonate and carboxylate groups. More preferred anionic substitutents are sulfonate groups. A preferred aromatic compound is a substituted or unsubstituted polycyclic aromatic compound having at least one anionic substituent. Preferred polycyclic aromatic sulfonates are amaranth and calcion.